ABSTRACT Initiation of reverse transcription of genomic RNA is a critical step in retrovirus replication. In HIV-1, the primer for this reaction is human tRNALys3, which is selectively packaged into virions and annealed to the primer binding site (PBS) in the 5? untranslated region (UTR) of HIV-1 viral RNA (vRNA) to form the reverse transcription initiation complex (RTIC). While numerous structures of HIV-1 reverse transcriptase (RT) complexes with various substrates and inhibitors have been solved by Arnold and others, there have been no high-resolution structures of the RTIC, which is one of the most important missing snapshots of the virus life cycle. Another knowledge gap in the early events of viral infection is the structural mechanism by which members of the APOBEC3 (A3) family of restriction factors suppress retrovirus infection. Seminal work by project investigator Malim established that HIV-1 evades A3- mediated inhibition through the action of its Vif protein. It is also known that A3 proteins induce excessive cytidine-to-uridine editing of nascent HIV-1 cDNA leading to hypermutation and genetic inactivation. However, A3-mediated inhibition of HIV-1 also involves direct suppression of RT transcription itself, through a mechanism that is poorly understood. Moreover, structures of full-length A3G have not been obtained, despite intensive efforts. In Project 3, the Arnold (Core 4), Griffin (Core 2), Lyumkis (Core 1), Malim, Marcotrigiano (Core 3), Millar, Musier-Forsyth, Olson (Project 6), and Sarafianos groups will work together in a highly integrated and synergistic fashion to address these knowledge gaps in two specific aims. The first aim will focus on the characterization of RT interactions with tRNA and vRNA in the initiation complexes of HIV-1 and prototypic foamy virus (PFV), leveraging strong preliminary data from X-ray crystallography, cryo-electron microscopy (cryo-EM), hydrogen/deuterium exchange (HDX), small angle X-ray scattering (SAXS), and single molecule biophyiscal experiments. In the second aim, preliminary work demonstrated the ability to assemble stable RT-A3G complexes and will provide a platform that can be employed for the structural, biophysical, and virological characterization of RT interactions with A3 proteins, including the elusive full-length A3G. Such work will be central to illuminating an atomic understanding of both cytidine deamination and RT inhibition, and may be of relevance to other A3 proteins such as A3B whose causative role in cancer-associated mutations has recently emerged. Taken together, work in this Project will provide breakthroughs into the understanding of molecular processes that control early steps in retroviral infection.